Protein Extraction Conditions Research Articles

Feasibility of ultrasound-assisted optimized process of high purity rice bran protein extraction

ABSTRACT: Rice bran is a by-product of the rice milling process, found worldwide in abundance and highlighted due its protein content. This study optimized the conditions for ultrasonic-assisted extraction of protein from defatted rice bran (DRB) and characterized the rice bran protein concentrate (RBPC). A sequential strategy of experimental design was employed; the effect of pH, temperature, ultrasound exposure time, and amplitude were evaluated regarding the percentage of protein extraction by a Full factorial design (FFD) with a fixed frequencies (FFD-A: 37 kHz; FFD-B: 80 kHz). Subsequently, the percentage of protein extracted was optimized employing a Central composite rotatable design (variables: pH and ultrasound exposure time) and RBPC obtained was characterized regarding chemical and functional properties. The pH and ultrasound exposure time had positive effect (P ≤ 0.05) on percentage of protein extraction; moreover, 37 kHz frequency was more effective in protein extraction. The optimized condition (frequency: 37 kHz; temperature: 30 °C; pH: 10; ultrasound exposure time: 30 min; and amplitude: 100%) allowed 15.07% of protein recovery and the RBPC presented 84.76 g 100 g-1 of protein. Magnesium and copper were the main mineral in RBPC (34.4 and 25.5 µg g-1, respectively), while leucine was the limiting amino acid (0.42) and threonine presented the highest chemical score (1.0). The RBPC solubility was minimal at pH 4 and higher at pHs 6-10; the water and oil absorption capacity were higher than bovine serum albumin (BSA) and the emulsifying capacity was comparable to BSA, with a suitable stability. It was possible to obtain a higher purity RBPC than described in the literature, due to the optimization in the extraction process steps, with functional properties suitable for application in food products, especially emulsified ones.

Recovery of Protein from Dairy Milk Waste Product Using Alcohol-Salt Liquid Biphasic Flotation

Expired dairy products are often disposed of due to the potential health hazard they pose to living organisms. Lack of methods to recover valuable components from them are also a reason for manufactures to dispose of the expired dairy products. Milk encompasses several different components with their own functional properties that can be applied in production of food and non-food technical products. This study aims to investigate the novel approach of using liquid biphasic flotation (LBF) method for protein extraction from expired milk products and obtaining the optimal operating conditions for protein extraction. The optimized conditions were found at 80% concentration ethanol as top phase, 150 g/L dipotassium hydrogen phosphate along with 10% (w/v) milk as bottom phase, and a flotation time of 7.5 min. The protein recovery yield and separation efficiency after optimization were 94.97% and 86.289%, respectively. The experiment has been scaled up by 40 times to ensure it can be commercialized, and the protein recovery yield and separation efficiency were found to be 78.92% and 85.62%, respectively. This novel approach gives a chance for expired milk products to be changed from waste to raw materials which is beneficial for the environment and the economy.

Biorefining and the Functional Properties of Proteins from Lipid and Pigment Extract Residue of Chlorella pyrenoidosa

Microalgae are considered as excellent candidates for bioactive compounds, yet microalgal residues remaining after the extraction of one or two compounds are usually discarded, which is not economical. This study demonstrates the alkaline extraction of proteins from Chlorella pyrenoidosa residue after lipid and pigment extractions, and their functional properties. Single-factor experiments and response surface methodology were used to obtain the optimal conditions for protein extraction. Based on our results, a maximum protein yield of 722.70 mg/g, was obtained under the following extraction conditions: sodium hydroxide concentration 7.90%, extraction temperature 70.00 °C, extraction time 34.80 min, and microalgal residue concentration 8.20 mg/mL. The molecular weight of microalgal residue protein isolate (MRPI) was mainly distributed at the regions of 0.18–0.50 kDa, 0.50–1.50 kDa, and 1.50–5.00 kDa. The essential amino acid content was greater than the values recommended by FAO/WHO standards; a high essential amino acid index value (1.49) was another good indication that MRPI is suitable for human consumption. Moreover, MRPI exhibited excellent emulsifying properties and antioxidant activity, which suggests it may be useful as an emulsifying agent and antioxidant. These findings could improve the extraction methods of functional protein from microalgal residue and add value to microalgae-based bioactive compound production processes.

Recovery and Extraction of Technofunctional Proteins from Porcine Spleen Using Response Surface Methodology

Porcine spleen is an edible meat by-product from industrial slaughterhouses with a low commercial value that is generally underutilised. In this work, response surface methodology was used to optimise the conditions for protein extraction from porcine spleen. Factors examined were pH (4.3–8.6) and salt concentration (0–4%) of the extraction buffer. The response of several physicochemical characteristics and technofunctional properties of spleen protein fractions as a function of two particular controllable factors of the fractionation process was fitted to second-order polynomial models. The proximate composition (%) of porcine spleen was as follows: moisture (78.9 ± 0.4), protein (17.2 ± 0.7), fat (2.9 ± 0.4), total ash (1.34 ± 0.1) and hydroxyproline (0.20 ± 0.1). Fe content (mg/kg) was 275.5 ± 63. SDS-PAGE patterns of the spleen protein fractions revealed multiple bands with low and high molecular weights from 15 to 220 kDa, corresponding to sarcoplasmic, myofibrillar and connective tissue proteins in both soluble and insoluble fractions with slight differences due to pH and ionic strength extraction conditions. Significant second-order models were obtained for the response variables (protein solubility), foaming and emulsifying properties of soluble fraction, and redness (a*), chroma (C*) and retention properties -cooking loses and water holding capacity- of insoluble residue from porcine spleen. The analysis of the fitted model plots and the ANOVA confirmed that model fits were satisfactory.

Optimization of preliminary purification conditions for protein extraction from tea residues

Optimization of preliminary purification conditions for protein extraction from tea residues

Continuous extraction of proteins with a miniaturized electrical split-flow cell equipped with suspended splitters fabricated by dry film lamination

This article describes the development of an electrically driven Split-Flow Thin microsystem suitable for continuous-flow protein purification or concentration. We used a technology based on dry film resist, Ordyl SY 355, to realize 3D suspended structures in a microchannel (150 μm), including patterned Cr/Pt electrodes. The device developed for this research is the first electrical miniaturized dry film SPLITT system including suspended splitters and represents a good miniaturization effort of the ideal SPLITT cell, thus expanding the possibilities of realizing split-flow fractionation microfluidic network based on dry film technology. A suitable design and fabrication process for the realization of such a kind of device is reported. The purification/concentration performances of the system have been tested by infusing a Bovine Serum Albumin solution (0.1 wt percentage) through the inlets and evaluating the protein content at the outlets after the application of a pulsed electric potential. Experimental conditions have been changed during the testing phase, by varying the applied potential and the duty cycle of the square wave and the flow rate to find suitable conditions for protein extraction. The best-achieved purification/concentration rate was around 60%, measured as concentration difference between the two output channels normalized on the input concentration, thus confirming the viability of device for protein enrichment or purification for sample preparation. The device has a potential for integration in micro-TAS, thanks to its reduced dimensions, for applications involving protein analysis or bio-sensing in matrices including proteins interfering with the measurement.

Optimization of protein extraction from Gelidiella acerosa by carbohydrases using response surface methodology

SUMMARYIn this study, application of response surface methodology for enzymic pretreatment optimization of Gelidiella acerosa was investigated in order to improve the extraction of algal proteins using Viscozyme L and Celluclast 1.5L. The total protein, soluble proteins and reducing sugar recovery in the water‐soluble fraction were studied in relation to the hydrolysis time, type and concentration of the enzymes. Enzymatic digestion appeared to be an effective treatment for protein extraction. While enzyme hydrolysis by Celluclast 1.5L was able to facilitate the protein extraction, it was a relatively inefficient way to improve protein extraction yield, in comparison with Viscozyme L. The optimum conditions for protein extraction was found to be hydrolysis by 2.8 μL mL−1 of Viscozyme L for 12 h.

Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone-treated smooth muscle tissues for phos-tag SDS-PAGE.

Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.

Optimization of non-denaturing protein extraction conditions for plant PPR proteins.

Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non-denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pI, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pI/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.

Protein concentrates from defatted rice bran: preparation and characterization

Abstract The goal of this work is to determine the optimal conditions for the obtaining of protein concentrates from rice bran. The effects of defatting processes applied to this product in laboratory and in industrial scales were investigated. Through two experimental designs were performed, and production conditions to obtain the protein concentrates were chosen with values of protein contents average 59.9% and 57.1%, with protein yields of 61.6% and 30.7%. The concentrates produced with industrially defatted rice bran showed higher digestibility and increased thermal stability compared with the product obtained with laboratory defatted rice bran. Both concentrates show molecular weight proteins below 50 kDa. The morphology of the precipitated proteins, analyzed by scanning electron microscopy, there was a great difference in the size of particles, which form the wet precipitates. The differences presented by the concentrates can be due to defatting processes which raw materials were submitted. Thus, studying the protein extraction conditions and knowing its characteristics is very important for the industry, because food processing requires knowing the behavior of each compound during and after processes they will be submitted.

Optimization of Protein Extraction and Decoloration Conditions for Tea Residues

To optimize alkaline method for extracting proteins from tea residue (TR), the effect of extraction conditions on tea protein extraction rate (TPER) was investigated. Single factor experiment showed the extraction temperature 80 °C, extraction time 100 min, pH value 13 and liquid–solid ratio 40:1 as the optimal extraction conditions. The orthogonal test revealed that the maximum TPER reached 29.71% under the following optimal combination of conditions: extraction temperature 70 °C, extraction time 60 min, pH 12 and liquid–solid ratio 50:1. For optimizing the purification of tea residue proteins, isoelectric point precipitation (pI), ammonium sulfate precipitation (aS) and isoelectric point plus ammonium sulfate precipitation (iPAS) were compared. The result showed that the highest protein precipitation rate (PPR) was 89.70% which was generated by using iPAS. Furthermore, powdered activated carbon was chosen as the most suitable decolorant for the extracted proteins.

Optimisation of extraction conditions and thermal properties of protein from the Andean pseudocereal cañihua (Chenopodium pallidicaule Aellen)

SummaryResponse surface methodology (RSM) was used to optimise alkaline protein extraction from cañihua grain meal as to maximise protein extraction yield. Different factors (temperature, extraction time, solvent/meal ratio, pH and NaCl molar concentration) were screened, and their significant influence on the extracted protein yield was evaluated. The first four factors were selected and studied by RSM using a central composite design (CCD). The obtained model produced a satisfactory fitting of the results (R2 = 0.801). Optimal cañihua protein extraction conditions corresponded to a temperature of 21 °C, time extraction of 5 min, solvent/meal ratio of 37/1 (v/w) at pH 10 resulting in a protein yield of 80.4 ± 1.3%, which closely agree with the predicted value of 81.4%. Moreover, protein degradation was studied via differential scanning calorimetry (DSC) obtaining a denaturation temperature of 93.4 °C and an enthalpy value of 1.22 ± 0.05 J g−1. These results are interesting from a technological point to help in designing an optimal protein extraction process and cañihua food processing strategies.

Peanut protein extraction conditions strongly influence yield of allergens Ara h 1 and 2 and sensitivity of immunoassays.

The clinical importance of peanut (Arachis hypogaea) allergies demands standardized allergen extraction protocols. We determined the effectiveness of common extraction conditions (20 buffers, defatting reagents, extraction time/temperatures, processing, extraction repeats) on crude protein and Ara h 1 and 2 yields. Despite similar 1D-gel profiles, defatting with n-hexane resulted in significantly higher yields of crude protein, Ara h 1, and Ara h 2 than with diethyl ether. The yields were affected by the composition and pH of the extraction buffers and other conditions, but crude protein yield did not always correlate with Ara h 1 and 2 yields. Denaturants, reducing agents, acidic buffers, and thermal processing of peanuts perturbed allergen quantification in ELISAs, probably via exposure of additional epitopes. Allergen detection in 2D-Western blots with PBS resulted in greater sensitivity than with TBS or Tris. We recommend that allergen extraction conditions be selected based on the research question being investigated.

Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics.

Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.

Extracting Protein from Antarctic Krill (Euphausia superba)

ABSTRACTThe isoelectric solubilization/precipitation (ISP) process was applied to Antarctic krill protein extraction. The solubility of Antarctic krill proteins and their fluoride content were studied between pH 1.00–13.75. The previous krill protein extraction conditions using ISP were improved. Excess fluoride in Antarctic krill was removed using a water-washing method according to its solubility. To save water, a multistage countercurrent system was used to optimize the ISP method. The results showed that the solubility of Antarctic krill proteins reached a minimum at pH 4.5 and a maximum at pH 13.5, while the concentration of dissolved fluoride was relatively unchanged (16.78–20.50 mg/L), with pH 2.00–13.50. Water-washing 4 times could reduce fluoride 98.14 ± 0.01% from krill with about a 5.14 ± 0.21% protein loss. The reduction allows krill protein to meet the Chinese National Standard (GB 2762-2005) for fluoride. Meanwhile, the four-stage countercurrent system achieved almost the same high fluoride removal rate (98.58 ± 0.01%) using only 25% of the water.

Optimized Methodology for Alkaline and Enzyme‐Assisted Extraction of Protein from Sacha Inchi (Plukenetia volubilis) Kernel Cake

AbstractThe residue after oil extraction from sacha inchi (SI) presents a high protein content of ∼59% that can be further exploited to extract proteins. In this study, the protein extraction parameters for defatted SI cake meal (DSICM) were optimized using alkaline and enzyme‐assisted extractions. A central composed design (CCD) was used to optimize the protein yield for both methods. The obtained response surface models (RSM) produced a satisfactory fitting of the results for both extraction methods (R2 = 0.9609–0.9761). For the alkaline extraction method, the optimal SI protein extraction conditions corresponded to 54.2C, solvent/meal 42/1 (v/w) ratio, NaCl concentration of 1.65 M, pH 9.5 for 30 min and yielded 29.7% protein. For the enzyme‐assisted method, optimal extraction conditions corresponded to an enzyme concentration of 5.6%, 40.4 min extraction, solvent/meal 50/1 (v/w) ratio, pH 9.0 and 50C and yielded 44.7% protein and hydrolysis degree of 7.8%.Practical ApplicationsThe cake obtained after oil extraction from SI seed is an important source of protein. Thus, efforts should focus on the development of protein extraction processes from the cake to add value to this by‐product. Up to date, studies are very limited. Results obtained in this study allowed the optimization of the protein extraction process from SI cake meal. The enzyme‐assisted protein extraction resulted in a higher quantity of protein recovery (∼1.5 fold more) than the alkaline protein extraction. The optimized protein extraction process will allow the food industry to obtain isolates or protein concentrates from SI cake meal to be used as techno‐functional, nutritional and/or functional agent.

Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH.

The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60, 90 and 120 °C for 30 min led to protein denaturation and aggregation, which was enhanced at higher treatment temperatures. The in vitro gastric digestibility measured during 6 h was lower for protein extracts pre-treated at 90 and 120 °C compared to 60 °C. The digestibility decreased with increasing extraction pH, which could be ascribed to protein aggregation. Protein digestibility of the quinoa protein isolates was higher compared to wholemeal quinoa flour. We conclude that an interactive effect of processing temperature and extraction pH on in vitro gastric digestibility of quinoa protein isolates obtained at various extraction pH is observed. This gives a first indication of how the nutritional value of quinoa protein could be influenced by heat processing, protein extraction conditions and other grain components.

Techno-economical evaluation of protein extraction for microalgae biorefinery

Due to scarcity of fossil feedstocks, there is an increasing demand for biobased fuels. Microalgae are considered as promising biobased feedstocks. However, microalgae based fuels are not yet produced at large scale at present. Applying biorefinery, not only for oil, but also for other components, such as carbohydrates and protein, may lead to the sustainable and economical microalgae-based fuels. This paper discusses two relatively mild conditions for microalgal protein extraction, based on alkali and enzymes. Green microalgae (Chlorella fusca) with and without prior lipid removal were used as feedstocks. Under mild conditions, more protein could be extracted using proteases, with the highest yields for microalgae meal (without lipids). The data on protein extraction yields were used to calculate the costs for producing 1 ton of microalgal protein. The processing cost for the alkaline method was € 2448 /ton protein. Enzymatic method performed better from an economic point of view with € 1367 /ton protein on processing costs. However, this is still far from industrially feasible. For both extraction methods, biomass cost per ton of produced product were high. A higher protein extraction yield can partially solve this problem, lowering processing cost to €620 and 1180 /ton protein product, using alkali and enzyme, respectively. Although alkaline method has lower processing cost, optimization appears to be better achievable using enzymes. If the enzymatic method can be optimized by lowering the amount of alkali added, leading to processing cost of € 633/ton protein product. Higher revenue can be generated when the residue after protein extraction can be sold as fuel, or better as a highly digestible feed for cattle.

Optimised methodology for the extraction of protein from quinoa (Chenopodium quinoa Willd.)

SummaryQuinoa is a highly appreciated Andean pseudo‐cereal and has sparked attention worldwide due to its excellent nutritional value. The protein extraction parameters for defatted quinoa seed meal (DQSM) were optimised in this study. Initially, a Plackett–Burman design was applied to screen the factors displaying a potential effect on the quinoa protein extraction yield (Y %, g soluble protein/100 g total protein) being the main evaluated factors: pH, NaCl concentration, time, temperature, solvent type, particle size and solvent/meal ratio. Four main factors: temperature, solvent/meal ratio, pH and time selected from the screening step were optimised with a central composed design (CCD). The obtained response surface model (RSM) produced a satisfactory fitting of the results (R2 = 0.9308). Optimal quinoa protein extraction conditions of 36.2 °C, solvent/meal ratio of 19.6/1 (v/w) and 90 min resulted in a protein yield of 62.1% (9.06 g of protein/100 g DQSM) which closely agree with the predicted value of 62.5%.The model was experimentally validated by extracting the quinoa protein using the optimal conditions revealed by RSM. The optimised conditions could be successfully employed in the design process of protein extraction from quinoa seed meal to obtain optimal yields.

Single unit filter-aided method for fast proteomic analysis of tear fluid

Human tear fluid is a complex mixture containing high concentrations of proteins and is increasingly becoming an important source for studying protein biomarkers of eye-related diseases such as Graves’ ophthalmopathy. Today, the Schirmer tear test is the most widely used technique for tear collection. However, sample handling and protein extraction from these strips have been highly challenging. Cutting and removal of the Schirmer strips after extraction, which may lead to sample loss prior to downstream analysis, are some of the challenges to consider. To address some of these limitations, we have developed a single-unit filter-aided method for both sample handling and protein extraction. In addition, we systematically investigated the most suitable conditions for protein extraction from these strips. Among the different extraction conditions applied, extraction with 100mM ammonium bicarbonate containing 50mM NaCl resulted in the highest number of identified proteins using one-dimensional liquid chromatography tandem mass spectrometry (LC–MS/MS). Moreover, 1526 proteins were identified when the optimized extraction method was combined with two-dimensional LC–MS/MS analysis, demonstrating the applicability of this novel approach to the study of the tear proteome. This dataset of identified proteins represents a comprehensive catalogue of the tear proteome and may serve as a list for future biomarker research.